The prognosis of HPV+ oropharyngeal cancer (OPC) treated with standard radiation at 70 Gy is excellent. However, 80% of these patients experience grade ?2 mucositis and 30% have permanent swallowing dysfunction. Clinical data suggest that 70 Gy may be overtreatment for some HPV+ OPCs. A modest reduction of 10-16 Gy for an unselected cohort with HPV+ OPC showed a 2-year progression-free survival (PFS) of 80%, but 40% of patients still had difficulty swallowing at 1 year. The proposed research will employ imaging (PET/MRI) biomarkers to identify patients with HPV+ OPC who may benefit from a major dose reduction to 30 Gy, a dose based on experience in HPV+ anal cancer, with the goal of maintaining tumor control and cure while substantially reducing treatment-related toxicity. A pilot trial of 19 HPV+ OPC patients treated at 30 Gy followed by neck dissection was encouraging, with a 2-year PFS of 93%. Significant toxicity reduction was observed. The proposed research will expand on the initial findings of the proof- of-principle study to a larger cohort of patients. The proposed imaging metrics to select patients for major dose de- escalation will include baseline and early intra-treatment [18F]-FMISO PET imaging, which will provide information on tumor hypoxia, a marker of radioresistance (Aim 1). Eligible patients will have no evidence of hypoxia on baseline imaging or have resolution of hypoxia during treatment, which will portend tumor radiosensitivity. We will interrogate the tumor microenvironment (Aim 2) by deriving quantitative imaging biomarkers (QIBs) from multi-parametric diffusion-weighted MRI (DW-MRI) consisting of non-Gaussian intravoxel incoherent motion (NG-IVIM) as well as [18F]-FMISO) PET imaging to select appropriate 30 Gy candidates to avoid neck dissection, with the goal of further toxicity reduction. The change in intra- treatment diffusion (D, surrogate of tumor cellularity) and kurtosis (K, surrogate of tissue microstructure) from baseline DW-MRI will guide which patients de-escalated to 30 Gy can avoid neck dissection. HPV is known to dysregulate the DNA damage response (DDR) and double-strand break (DSB) repair pathways to facilitate viral replication. Preclinical work suggests that this dysregulation accounts for the radiosensitivity of HPV+ OPC, although there are conflicting data regarding the precise nature of the responsible defect. For Aim 3, whole-genome sequencing (WGS) with mutational signature analyses will be used to identify DDR and DSB repair defects in individual HPV tumors and characterize the clinical influence on radiosensitivity. The relationship between genomic signatures and non-invasive imaging of tumor hypoxia and tumor cellularity that portend radiobiological sensitivity also will be explored. The proposal's central hypothesis is that PET/MRI of HPV+ OPC classification with the underpinnings of a molecular characterization of the cancer biology will yield a robust decision tool to stratify patients for whom dose de-escalation to 30 Gy will provide a clinical benefit and significantly reduced toxicity, without compromising treatment outcome.